PROJECT SUMMARY Pancreatic cancer (PDAC), characterized by its aggressive nature and extensive fibrosis within the tumor microenvironment (TME), has a dismal 5-year survival rate of only 7%. Pancreatic tumors consist of abundant extracellular matrix and tumor-associated fibroblasts (TAFs) that express fibroblast activation protein alpha (FAP). FAP+TAFs promote tumor development and progression in the pancreas through remodeling the extracellular matrix and suppressing local CD8+ T cell response during carcinogenesis. Pancreatic tumors contain FAP+TAFs; the degree of FAP expression positively correlates with lymph node status, larger tumor size, faster recurrence, and worse clinical outcome. Since FAP is overexpressed in tumor tissue and has basal expression in benign tissue, it has value as a target for cancer therapies that modulate tumor-stroma interactions. However, quantifying FAP expression in vivo with available molecular probes is difficult, and a key unanswered question is whether PET-based molecular imaging agents can detect FAP+TAFs in the PDAC tumor stroma. In this application we hypothesize that targeting fibroblast activation protein alpha (FAP) ? a unique biomarker found on tumor-associated fibroblasts (TAFs) in the PDAC TME, will allow us to detect and quantify the TME in vivo using PET. To test this hypothesis, we propose two specific aims. In the first aim we will synthesize novel FAP inhibitors that are based upon a cyanopyrrolidine scaffold. These molecules will be assessed for FAP selectivity using plate-based IC50 and affinity assays, lipophilicity (log P) and in vitro stability using human liver microsomes. In Aim 2 the most promising compounds will be radiolabeled with carbon-11 and evaluated in biodistribution and small animal PET/CT studies using a PDX orthotopic xenograft mouse model, which recapitulates the heterogeneity of PDAC tumors observed in the clinic. Radioactivity accumulation will be quantified from regions of interest (ROIs) drawn around the tumor on the co-registered PET/CT images. Finally, multi-spectral microscopy will be conducted on murine tissue specimens to assess radiotracer specificity and correlate the radioactivity accumulation with FAP and other biomarkers that describe a TAF phenotype. If successful, this work will immediately impact how basic scientists study FAP expression and the pancreatic TME. This work could inform new paradigms for pancreatic cancer imaging that can be exploited as part of an integrated strategy for much-needed improvements in diagnosis and management of pancreatic carcinoma.